Preparation Of Two Monoclonal Antibodies And Establishment Of Enzyme-linked Immunosorbent Assay Method For Florfenicol And Thiamphenicol

Florfenicol(FF)and thiamphenicol(TAP)are broad spectrum antibiotics of chloramphenicol permitted for the treatment of respiratory and intestinal infections in cattle,poultry and swine,Chloramphenicol(CAP)is harmful to human,so it has been banned around the world.While FF and TAP are less toxic than CAP,it has been reported that TAP has strong immunosuppressive effects,and FF has embryo toxicity which is not permitted in pregnant animals.Consequently the maximum residue limits(MRLs)for FF and TAP were established in the China,US,EU,Japan and many other countries and organizations.Currently,instrumental methods have been able to achieve the testing requirements of the two drugs.Although instrumental methods have high accuracy and precision,and they often require complex sample cleanup and sophisticated laboratory equipment as well as highly trained operators.Then instrumental methods are unsuitable for high sampling frequency or rapid assessment of results.ELISA method is a sensitive.accurate and efficient method which is suit for screening large numbers of samples..In this study,by the designing the haptens,synthesizing the antigens and utilizing monoclonal antibody preparation technology,we obtained two hybridomas lines recognizing FF and TAP,and a competitive indirect enzyme-linked immunosorbent assay that could detect FF and TAP in swine,chicken,swine liver,chicken liver and fish at relevant levels was developed.The main results were as follows:1.FF as a raw material to synthesize hapten FF-HS,it was identified successfully by mass spectrometry.FF-HS was linked to keyhole limpet(KLH),albumin human serum(HSA)and ovalbumin(OVA)using DCC as cross-linker.These antigens were identified by UV spectrum;FFA was linked to KLH,HSA and OVA using EDC or GA as cross-linker respectively.These antigens were identified by UV spectrum;FFA as a raw material to synthesize hapten FFA-HS,it was identified by mass spectrometry.FFA-HS was linked HSA and OVA by using DCC as cross-linker.These antigens were identified by UV spectrum.2.Female Balb/c mice were immunized with the antigens mentioned above.Through the heterologous and homologous assay format,the antisera titers and specificity of mice were monitored by ELISA.It was best when useing FF-HS-KLH/HSA and FFA-EDC-OVA,the antisera colud identify florfenicol and thiamphenicol.The antisera from FFA-EDC-KLH/HSA and FFA-GA-KLH/HSA had high titers but no specificity.The antisera from FFA-HS-HSA had low titers and no specificity yet.By monoclonal antibody technology,we obtained two hybridomas lines named FF/7A8 and TAP/5F4.3.The average chromosome number of the monoclonal hybridoma line FF/7A8 was about 102.8.By ascites tumor,we prepared monoclonal antibody,and the MAb had a IgG1 subclass.A ciELISA for detecting the residue of FF was established by optimizing conditions.The results showed that the best coating concentration of FF/7A8 MAb was 1 ?g/mL,and the best dilution was 1:240000.A good linearity was achieved over a concentration range of 0.05 to 0.8 ?g/L,and IC50 value was 0.21±0.02 ?g/L.The inter-assay and intra-assay coefficient of variation were below 10%.The sensitivity of ciELISA was 0.037 ?g/L.4.The average chromosome number of the monoclonal hybridoma line TAP/5F4 was about 104.5.By ascites tumor,we prepared monoclonal antibody,and the MAb had a IgG1 subclass.A ciELISA for detecting the residue of TAP was established by optimizing conditions.The results showed that the best coating concentration of TAP/5F4 MAb was 1 ?g/mL,and the best dilution MAb was 1:16000.A good linearity was achieved over a concentration range of 0.1 to 1.6 ?g/L,and IC50 value was 0.35±0.03 ?g/L.The inter-assay and intra-assay coefficient of variation were both below 10%.The sensitivity of ciELISA was 0.065 ?g/L.The antiboy had good specificity for both FF and TAP,the cross-reactivy about FF is 167%.5.For a spiking study of swine,chicken,swine liver,chicken liver and fish,samples were spiked with FF at three different levels(0.15,0.3 and 0.6 ?g/kg).We carried out rapid extraction methods with ethyl acetate,and the recoveries of spiked samples were 78.0%?111.8%.The inter-assay and intra-assay coefficient of variation were both below 15%,indicating the ELISA method had good accuracy and reproducibility.6.For a spiking study of swine,chicken,swine liver,chicken liver and fish,samples were spiked with FF and TAP respectively.FF at three different levels(0.15,0.3 and 0.6?g/kg)and TAP at three different levels(0.2,0.4 and 0.8 ?g/kg).We developed rapid extraction methods with ethyl acetate,and the recoveries of spiked samples of FF were 72.8%?110.5%.The inter-assay and intra-assay coefficient of variation were less than 15%;and the recoveries of spiked samples of TAP were 75.3%?113.4%.The inter-assay and intra-assay coefficient of variation were below 15%.The results indicated the ELISA method had good accuracy and reproducibility.7.Compared with LC-MS/MS,the ELISA results correlated very well,indicating the reliability of the test we developed.In this study we obtained two antibodies,which could detect FF and TAP and the sensitivity and specificity were better than that of similar products at home and abroad.We established the extraction method for FF and TAP in edible animal tissues,and the method was simple,fast and sensitive.We established a ELISA detection methods with high sensitivity and specificity for FF and TAP,it had a high academic value and broad application prospect.